Genomic instability in long-term culture of human adipose-derived mesenchymal stromal cells.

Mesenchymal stromal/stem cells stem (MSC) have been widely studied due to their great potential for application in tissue engineering and regenerative and translational medicine. In MSC-based therapy for human diseases, cell proliferation is required to obtain a large and adequate number of cells to ensure therapeutic efficacy. During in vitro culture, cells are under an artificial environment and manipulative stress that can affect genetic stability. Several regulatory agencies have established guidelines to ensure greater safety in cell-based regenerative and translational medicine, but there is no specific definition about the maximum number of passages that ensure the lowest possible risk in MSC-based regenerative medicine. In this context, the aim of this study was to analyze DNA damage and chromosome alterations in adipose-derived mesenchymal stromal cells (ADMSC) until the eleventh passage and to provide additional subsidies to regulatory agencies related to number of passages in these cells. Thus, two methods in genetic toxicology were adopted: comet assay and micronucleus test. The comet assay results showed an increase in DNA damage from the fifth passage onwards. The micronucleus test showed a statistically significant increase of micronucleus from the seventh passage onwards, indicating a possible mutagenic effect associated with the increase in the number of passages. Based on these results, it is important to emphasize the need to assess genetic toxicology and inclusion of new guidelines by regulatory agencies to guarantee the safety of MSC-based therapies for human diseases.

Genomic instability in long-term culture of human adipose-derived mesenchymal stromal cells

Mesenchymal stromal/stem cells stem (MSC) have been widely studied due to their great potential for application in tissue engineering and regenerative and translational medicine. In MSC-based therapy for human diseases, cell proliferation is required to obtain a large and adequate number of cells to ensure therapeutic efficacy. During in vitro culture, cells are under an artificial environment and manipulative stress that can affect genetic stability. Several regulatory agencies have established guidelines to ensure greater safety in cell-based regenerative and translational medicine, but there is no specific definition about the maximum number of passages that ensure the lowest possible risk in MSC-based regenerative medicine. In this context, the aim of this study was to analyze DNA damage and chromosome alterations in adipose-derived mesenchymal stromal cells (ADMSC) until the eleventh passage and to provide additional subsidies to regulatory agencies related to number of passages in these cells. Thus, two methods in genetic toxicology were adopted: comet assay and micronucleus test. The comet assay results showed an increase in DNA damage from the fifth passage onwards. The micronucleus test showed a statistically significant increase of micronucleus from the seventh passage onwards, indicating a possible mutagenic effect associated with the increase in the number of passages. Based on these results, it is important to emphasize the need to assess genetic toxicology and inclusion of new guidelines by regulatory agencies to guarantee the safety of MSC-based therapies for human diseases.

[Activity of daptomycin, ciprofloxacin, clindamycin and cotrimoxazole against coagulase-negative Staphylococcus strains with diminished susceptibility to vancomycin]. / Actividad de daptomicina, ciprofloxacino, clindamicina y cotrimoxazol frente a diferentes cepas de Staphylococcus coagulasa negativo con sensibilidad conservada y disminuida para vancomicina.

INTRODUCTION: Coagulase Negative Staphylococci (CNS) have become one of the most common nosocomial pathogens and it has a high mortality rate due to the increased of seriously ill patients survival, long states immunosuppression and presence of foreign bodies, such as catheters, prostheses, pacemakers, etc. In addition, there is a significant increase in resistance to antimicrobial drugs, especially beta-lactams, and the increase in the MIC for vancomycin leads to a loss of clinical efficacy. This necessitates the search for new therapeutic alternatives, such as daptomycin. The aim of this paper is to study the activity of daptomycin, ciprofloxacin, clindamycin and cotrimoxazole in two groups of clinically significant CNS: a MIC90with vancomycin ≤ 1 mg/L and the other with MIC90 2 mg/L. METHODS: We identified and studied MIC90 to ciprofloxacin, clindamycin and cotrimoxazole from 54 strains of clinically significant by the CNS Combo 22 Microscan panels (Dade Behring, Siemens). The MIC90for daptomycin was performed using Etest (AB BioMérieux, Solna, Sweden) on Mueller Hinton plates (BioMérieux, France). RESULTS: In Group I (vancomycin MIC90 ≤ 1 mg/L) were 19 strains whereas in Group II (vancomycin MIC90 =2 mg/L) were 35 strains. Expressed in mg/L, MIC90 ranges for daptomycin were 0.047-0.5 in Group I and 0.064-0.5 in Group II. for ciprofloxacin were 8 sensitive strains and 11 resistant in Group I and 10 sensitive and 25 resistant in Group II. For clindamycin were 7 sensitive strains and 12 resistant in Group I and 16 sensitive and 19 resistant in Group II. Finally, for cotrimoxazole were 10 sensitive strains and 9 resistant in Group I and 19 sensitive and 16 resistant in Group II. CONCLUSIONS: The MIC levels to daptomycin were not influenced by the increase in the MIC for vancomycin. There was no statistically significant difference for the sensitivity of ciprofloxacin between the two groups of vancomycin. Regardless of vancomycin, there were a clear relationship between the sensitivity of ciprofloxacin with clindamycin and cotrimoxazole.

Investigation of taxol as a potential radiation sensitizer.

BACKGROUND: The authors evaluated the effects of taxol, a microtubular inhibitor, as a possible radiation sensitizer on the human leukemic cell line (HL-60). Taxol acts as a mitotic inhibitor, blocking cells in the G2M-phase of the cell cycle. The differential radiation sensitivity of cells in various phases of the cell cycle has been well recognized. This study was focused on the possible interaction between radiation and a microtubular inhibitor, taxol, in regard to its ability to synchronize cells at the G2M-phase of cell cycle and, thereby, enhance the radiation sensitivity of the cells. METHODS: HL-60 cells were exposed to 3 x 10(-8) M concentrations of taxol for 1 hour at 37 degrees C followed by reculturing for 24 hours in drug-free medium. The cells were then seeded into 60-mm diameter plastic dishes at appropriate cell concentrations to estimate their colony-forming efficiency. The radiation dose ranged from 0-400 cGy and was delivered in a single fraction. The cellular survival after treatment with the drug and/or radiation was determined using a soft agar clonogenic assay. RESULTS: When HL-60 cells were treated with taxol, up to 70% of the cells were blocked in G2M-phase, as determined by flow cytometric analysis. At the low cytotoxic dose of 3 X 10(-8) M, the sensitizing enhancement ratio was 1.48. CONCLUSIONS: It appears that taxol has a radiation-sensitizing effect on HL-60 cells and deserves further investigation with other cell lines.